Gene of human epidermal growth factor receptor 2, HER2, also known as ERBB2 or Neu, is located on chromosome 17, and shows amplification in 20% to 30% of all female breast cancers. Amplification of HER2 has been identified as a prognostic and predicative marker and also a therapeutic target for an anticancer drug, Trastuzumab (Herceptin). Many studies also have demonstrated the importance of HER2 as a marker to other types of cancers such as bladder cancer, non-small cell lung cancer, ovarian cancer, bone cancer, head and neck cancer, pancreatic cancer, prostate cancer and stomach cancer. A recent clinical trial sponsored by Roche found that the addition of Herceptin to chemotherapy increase overall survival in HER2-positive advanced stomach cancer.
Before treatment with Herceptin, it is important to determine the HER2 status accurately, because only the patients with HER2 over-expression and/or amplification benefit from the treatment. The two most widely adopted testing methods for HER2 status are immunohistochemistry (IHC) that tests HER2 protein level, and fluorescence in situ hybridization (FISH) that tests HER2 gene copies. A great advantage of these two techniques is their convenience for use on the formalin-fixed paraffin embedded tissue (FFPE), a major form of archival material. However, there are documented and published disadvantages of using IHC and FISH. The scientific disadvantages of IHC are: (1) results may be affected by tissue fixation and processing methods; (2) no standardized scoring systems exists, and disparate scoring systems have been employed, for example, some take into consideration the proportion of positive tumor cells, some only regard the intensity of staining, and other combine the two parameters into one index.
Internationally, the algorithm for HER2 testing is to perform IHC to assess HER2 over-expression, in which patients with equivocal HER2 expression (2+) are further tested to assess HER2 amplification using FISH method, or patients are initially assessed for HER2 status by FISH method. FISH methods approved by US Food and Drug Administration (US-FDA) for predicting the effect of Herceptin therapy depend on the ratio between HER2 gene and chromosome 17, which distinguish HER2 gene amplification from chromosome 17 aneusomy.
The scientific disadvantages of FISH are: (1) possible HER2 over-expression by single gene not detectable, (2) difficulties associated with identification of invasive tissue (hard to identify the correct cells to count due to the lack of conventional morphologic features in the tissue), (3) difficult interpretation of borderline amplification, (4) temporary nature of staining due to the fluorescence signal fades over time. From the cost and benefit point of view, FISH is more expensive than IHC, because of the need for a fluorescence microscope, high cost of reagents, and lengthy processing time. CISH (Chromogenic In situ Hybridization) and SISH (Silver In situ Hybridization) are two emerging techniques which also measure gene copies in tumor cells.
Real-time or quantitative PCR (Polymerase Chain Reaction) has emerged as a superior alternative to the existing HER2 testing methods because of its high accuracy, wide dynamic range, and easy operation. Real-time PCR, a technology that is originally designed to measure mRNA expression level in cells, has been applied in detecting infectious diseases such as AIDS and SARS, and begins to make its way into the molecular diagnostics arena. There are some PCR-based test kits approved by US-FDA and seen to be used in ‘home brew’ or CLIA (Clinical Laboratory Improvement Amendments) certified laboratories. One good example is Oncotype Dx test which was approved by FDA under CLIA to perform PCR test for a number of breast cancer genes including HER2 at Genomic Health reference lab. There is no known FDA-approved HER2 PCR test kit for general clinical use at this time.